Progressive fibrotic diseases of the liver are a major cause of death throughout the world. The pathogenic process of fibrosis in the liver is critically dependent on proliferation and activation of hepatic stellate cells (also called lipocytes, or fat-storing or Ito cells) and other liver extracellular matrix-producing cells (i.e. portal myofibroblasts and fibroblasts), which synthesize and secrete excess extracellular matrix proteins (1). This process is common to liver disease of all etiologies. Chronic viral hepatitis B and C, alcoholic liver disease, non-alcoholic fatty liver disease and autoimmune and genetic liver diseases all entail the common final pathway of progressive liver fibrosis and the eventual development of cirrhosis.
Hepatic fibrosis is a reversible accumulation of extracellular matrix in response to chronic injury in which nodules have not yet developed, whereas cirrhosis implies a clinically important stage in this process that is usually but not always irreversible, in which thick bands of matrix fully encircle the parenchyma, forming nodules. Cirrhosis is associated with increased risks of liver failure, liver cancer and death. Consequently, to be effective, any liver disease therapy must be directed towards patients with reversible disease (fibrosis), which requires early identification and monitoring of those at risk (2).
Diagnosis of liver fibrosis is usually made by the histological analysis of liver biopsies. A single biopsy can be highly informative in determining diagnosis, prognosis and appropriate management (1A; 2A). The role of surrogate markers in the detection of liver fibrosis is not yet established. Accordingly liver biopsy is currently regarded as the “reference-standard” index of liver fibrosis.
Obtaining biopsies however is costly (3A) and is associated with pain (4A), hemorrhage, or death (5A; 6A). Processing of biopsies is time consuming and labor intensive. For all these reasons frequent repetition of liver biopsies is deemed unacceptable to patients and doctors alike, although monitoring the evolution of disease or response to treatment may require repeated biopsies.
Due to the small size of a needle biopsy and the diffuse nature of many liver diseases, biopsies may not yield results that are truly representative of a patient's disease status (7A). The histological analysis of biopsies requires experience and skill, but remains subjective and prone to both intra- and inter-observer variation (8A; 9A).
There is a considerable clinical need to identify surrogate markers of liver fibrosis. Such markers could be used to estimate the extent of fibrosis in place of a biopsy or, alternatively, they could be used in conjunction with a single liver biopsy to follow-up progression or regression of fibrosis and response to changes in life-style, or anti-fibrotic, antiviral, or other therapies. Ideally, such markers would be based on accurate and reproducible tests that could be automated and performed repeatedly with little disruption to patients.
Serum assays for products of matrix synthesis or degradation, and the enzymes involved in these processes, have been investigated as surrogate markers of liver fibrosis in a number of studies (10A-19A). Generally, the diagnostic performance of these markers has been disappointing, although some of individual assays have shown promise in detecting cirrhosis (20A; 21A), in alcoholic liver disease (Hyaluronic Acid) (22A), or milder fibrosis in non-alcoholic fatty liver disease (NAFLD) (YKL-40) (23A). Other markers have been reported to reflect changes in liver histology attributable to antiviral therapy (11A; 24A; 25A).
Biopsy and the serum markers compare different things: serum parameters characterize dynamic processes in the liver, while the biopsy characterizes the fibrotic manifestation at a fixed time-point. There may be a highly active fibrotic process in the liver, although fibrotic tissue has not yet been developed. In contrast, there may be large clusters of fibrotic tissue in the liver but the fibrotic activity stopped or discontinued temporarily.
An alternative approach is to combine a number of serum markers to generate an algorithm capable of evaluating fibrosis over a range of severity. In chronic Hepatitis C (CHC) (18A; 26A), and chronic hepatitis B, five parameters have been identified that could detect significant fibrosis with a positive predictive value (PPV) of 80%. However, these approaches failed to determine the severity of fibrosis in approximately 50% of patients and subsequent independent validation has questioned the utility of these markers (Rossi, et al., Clinical Chemistry. 49(3):450-4, 2003 March).
Previous studies have suggested that serum levels of extracellular matrix proteins (or their cleavage fragments) may be used to assess the severity and progression of liver fibrosis (U.S. Pat. No. 5,316,914, and EP 0 283 779). Different serum markers have been investigated and correlations with liver biopsies and severity of liver diseases have been found (6). Some of the makers that have been used for the assessment of liver fibrosis are PIIINP, Laminin, Hyaluronan, Collagen IV, TIMP-1, Tenascin, MMP-2 and Fibronectin. These markers have been measured and their capability to assess liver fibrosis has been shown. Nevertheless, neither the diagnostic accuracy nor the specificity of diagnostic markers is adequate to predict fibrosis scores with sufficient clinical utility.
Combinations of markers have been used in an effort to increase the diagnostic value of the simple biological index PGA (which includes Prothrombin time (PT), serum gamma-glutamyl transpeptidase (GGT), apolipoprotein A1 (Apo A1)), and the index PGAA (which adds alpha-2-macroglobulin (A2M) to the PGA index) have been described for the diagnosis of alcoholic liver disease in drinkers (7, 8). Although the PGA and PGAA indices have been combined with single serum markers (9, 10), such serum markers have not yet provided a reliable means of assessing liver diseases.
More recently, α2-macroglobulin, α2-globulin (or haptoglobin), γ-globulin, apolipoprotein-A1, γ-glutamyl-trans-peptidase, and total bilirubin have been combined to assess the status of liver fibrosis (11). The marker algorithm derived showed a strong diagnostic performance at the very end of the fibrosis spectrum—either for the identification or for the exclusion of severe or relatively mild fibrosis. The algorithm did not provide a diagnostic tool useful for identifying patients with moderate degrees of fibrosis.
Pilette, et al., J. Hepatol., Vol. 28, No. 3, 1998, pages 439-446 (Chemical Abstracts, Vol. 130, No. 7, Feb. 15, 1999 (Columbus, Ohio, U.S.; abstract no. 78389)) (“Pilette”) disclosed the correlation of the diagnostic markers hyaluronate, N-terminal peptide of procollagen Ill, laminin, and other serum markers by a mathematical algorithm for purposes of histopathological evaluation of liver fibrosis. Pilette determined the best morphometric method for the evaluation of hepatic fibrosis but did not combine markers algorithmically to obtain a diagnostic systems or methods that were superior to those which only used hyaluronic acid.
Guechot, et al., Clinical Chemistry, Vol. 42, No. 4 (April 1996) pp. 558-563 (XP002 1 49459 Winston; U.S.) (“Guechot”), provided a comparative assessment of the performance of hyaluronic acid and PIIINP as serum markers to assess liver disease. However, Guechot made no attempt to combine the results from hyaluronic acid and PIIINP in order to obtain a serum marker-based assessment of liver fibrosis that would be superior to the use of any of the two markers alone.
Accordingly, the need exists for accurate, reproducible, and computer-implementable methods, systems, kits, and media that employ two or more liver disease-related blood markers, e.g., plasma or serum markers, to aid in the determination of the status or progress of a liver disease in a patient. Such methods, systems, kits, and media would enable health care providers to ascertain the status or progress of a patient's liver disease at two or more time points without subjecting the patient to risky biopsies.
Further, such methods, systems, kits, and media would prove useful in designing or monitoring liver-disease related clinical trials, and in screening for agents useful in the treatment of liver disease.